Question

You want to obtain the sequence of the gene that encodes a virus capsid protein. Since sequencing proteins is a complex process, you only have some fragments of protein sequence that appear below:

MDAALCSGIRGGW ……………………………………… ..YYSPGGVLETCKK

The information that appears corresponds to the amino and carboxyl terminal sequence of the protein, respectively. Design a procedure to clone the entire sequence by PCR. Pick the least degenerate primers in each case and justify the choice.

Could you have made a better choice of primers if we don't exactly want the entire protein sequence?

Answer 1

PCR is an in vitro DNA amplification method.
It utilizes specific primers that bind to the complementary regions in the template DNA.
Primers flank the sequence to be amplified. So, if we know the ends of the sequence, it is sufficient to amplify the entire flanking sequence (Provided that the sequence is within 10 kb length)

We have to reverse translate the given amino acid sequence to obtain the nucleic acid sequence.
From the nucleic acid sequence, we have to design degenerate primers.

Given sequence:
MDAALCSGIRGGW ………………………………………YYSPGGVLETCKK

The primer length should be ~20 bases. So, we will consider seven amino acids from either end.
Forward sequence: MDAALCS
>Forward primer
ATG G AY GCN GCN CTN TGY UCN

Reverse sequence: VLETCKK
Reverse primer:
YTT YTT RCA NGT YTC NAG NAC

Base codes:
Y = C or T
N = A, T, G, or C

R = A or G